The long-term objective of this research is to better understand the molecular functions of the Escherichia coli heat shock proteins, DnaK and GroELS. These proteins belong to a highly evolutionarily conserved class of molecules called chaperonins, which catalyze the folding of polypeptide chains into their final conformations. Protein folding catalysis has been shown to be important in a variety of fundamental cellular processes, such as membrane translocation, assembly of oligomeric protein complexes and protection of cellular components against stress-induced damage. Because of the presence of these heat shock genes in single copy, E. coli provides an excellent system to study these proteins. A traditional genetic approach will be taken and will include the generation of mutations in both dnaK and groELS which affect their chaperonin active. A screening procedure will be employed which exploits the newly discovered ability of DnaK and GroEL to facilitate the export of LamB-LacZ hybrid proteins from the cytoplasm. Cells which efficiently export these LacZ hybrids become phenotypically Lac-. Lac+ revertants, will be isolated and should represent mutants which no longer possess chaperonin activity. New dnaK and groELS mutants will be characterized by phenotypic comparison with known alleles and by DNA sequence analysis. The ability of DnaK and GroEL to associate with LamB-LacZ hybrid proteins in vivo will also be investigated. In addition, the role of DnaK and GroELS in export of other E. coli proteins will be addressed by depleting the cells of these chaperonins. New mutants should prove useful in determining the roles of these heat shock proteins in cellular processes, such as protein export. In addition, the Lac phenotype of lamB-lacZ fusion strains will be used to search for other proteins, from a variety of organisms, which have chaperonin activity. An understanding of the molecular functioning of the chaperonins should provide insights into a variety of cellular process. They may prove useful to engineer E . coli to assembly protein complexes of medically and agriculturally important protein complexes. In addition, proteins are also apparently major antigenic determinants in infectious immune responses and may be important in immune surveillance. The isolation of new chaperonin mutants from E. coli should prove valuable in understanding the diverse molecular functions of this important class of molecules.